Isatin-tethered halogen-containing acylhydrazone derivatives as monoamine oxidase inhibitor with neuroprotective effect

Sixteen isatin-based hydrazone derivatives (IS1–IS16) were synthesized and assessed for their ability to inhibit monoamine oxidases (MAOs). All the molecules showed improved inhibitory MAO-B activity compared to MAO-A. Compound IS7 most potently inhibited MAO-B with an IC50 value of 0.082 μM, followed by IS13 and IS6 (IC50 = 0.104 and 0.124 μM, respectively). Compound IS15 most potently inhibited MAO-A with an IC50 value of 1.852 μM, followed by IS3 (IC50 = 2.385 μM). Compound IS6 had the highest selectivity index (SI) value of 263.80, followed by IS7 and IS13 (233.85 and 212.57, respectively). In the kinetic study, the Ki values of IS6, IS7, and IS13 for MAO-B were 0.068 ± 0.022, 0.044 ± 0.002, and 0.061 ± 0.001 μM, respectively, and that of IS15 for MAO-A was 1.004 ± 0.171 μM, and the compounds were reversible-type inhibitors. The lead compounds were central nervous system (CNS) permeable, as per parallel artificial membrane permeability assay (PAMPA) test results. The lead compounds were examined for their cytotoxicity and potential neuroprotective benefits in hazardous lipopolysaccharide (LPS)-exposed SH-SY5Y neuroblastoma cells. Pre-treatment with lead compounds enhanced anti-oxidant levels (SOD, CAT, GSH, and GPx) and decreased ROS and pro-inflammatory cytokine (IL-6, TNF-alpha, and NF-kB) production in LPS-intoxicated SH-SY5Y cells. To confirm the promising effects of the compound, molecular docking, dynamics, and MM-GBSA binding energy were used to examine the molecular basis of the IS7-MAO-B interaction. Our findings indicate that lead compounds are potential therapeutic agents to treat neurological illnesses, such as Parkinson's disease.

The mechanism-based inhibitors, selegiline and rasagiline (both MAO-B inhibitors) and clorgyline (a MAO-A inhibitor), are among the isoform-specific inhibitors described.Both MAO isoforms are inhibited by pargyline, a different propargylamine molecule.Other notable irreversible MAO inhibitors include the nonspecific inhibitors phenelzine and tranylcypromine 16,17 .The MAO-A inhibitors, toloxatone and moclobemide, and the MAO-B inhibitor, safinamide, are well-known examples of isoform-specific reversible inhibitors [18][19][20] .Dry mouth, nausea, diarrhea, constipation, drowsiness, sleeplessness, dizziness, and light-headedness are the most frequently reported side effects of the current medications used for treatment.When using a patch, skin irritation may also develop at the patch site.The search for novel MAO-A and MAO-B inhibitors has extensively used a variety of heterocycle families as scaffolds, including pyrazolines, chromones, chalcones, xanthines, benzyloxy, thiazoles, coumarins, and their precursors, isatin congeners, thiazolidiniones, and betacarboline [21][22][23][24][25][26] .As a result, isatin was identified as an effective MAO inhibitor.
Isatin (Fig. 1) is an endogenous small molecule with an indole-containing moiety and exhibits a broad range of biological and pharmacological activities.It comprises a nitrogen atom at position 1 and two carbonyl groups at positions 2 and 3.It further contains two rings: a six-membered aromatic ring and a five-membered antiaromatic ring 27,28 .It is widely distributed in the body fluids and different tissues of mammals and occurs naturally in plants 29 .In addition to clinical studies on the anticancer medications Toceranib, Semaxinib, and Orantinib, the Food and Drug Administration (FDA) has approved isatin-based therapies, such as Sunitinib (anti-tumor) and Nintedanib (anti-tumor) [30][31][32] (Fig. 1).
Isatin reversibly inhibits human MAO-A and MAO-B, with K i values of 15 and 3 µM, respectively 33 .According to previous studies, isatin is located close to the FAD cofactor in the MAO-B substrate cavity.The entrance cavity of the enzyme is free as isatin binds to its substrate cavity 34,35 .We hypothesized that the C-3 position could be exploited with hydrophobic moieties to improve MAO efficacy 26 .Therefore, we selected the C-3 position and replaced it with an acyl hydrazone linker and a halogenated phenyl (hydrophobic) moiety.The structural cores of acyl hydrazones, which include two distinctly connected nitrogen atoms, are generally responsible for the physical and chemical properties of these compounds.Therefore, acyl hydrazones are frequently used to develop novel molecules with various functions.Hydrazone derivatives have also been linked to MAO inhibition.Recently, Vishnu et al. synthesized piperonylic hydrazone-based isatin derivatives, however, insignificant interactions were observed in 3,4-methylenedioxy groups with MAO-B binding pocket 36 .Therefore, we replaced piperonylic with phenyl moiety and designed new approach toward acylhydrazone-based isatin derivatives (Fig. 2) to get a new family of effective MAO inhibitors in this study.

Synthesis
Benzoic acid (1 eq.) and hydrazine hydrate (2.5 eq.) were combined, and the reaction was conducted by using a microwave synthesizer (Monowave 50 Synthesizer, Anton-Paar, Graz, Austria) at 200 °C for 10-20 min.Upon completion of the reaction, the product benzohydrazide was recrystallized from methanol.Then, the mixture of isatin or substituted isatin (0.001 mol) and benzohydrazide (0.001 mol) 37,38 in methanol, by adding a catalytic amount of acetic acid, was placed in a reaction vial and subjected to the microwave synthesizer at 100-120 °C for 5-10 min.The reaction progress was monitored by using thin-layer chromatography (TLC) with an eluent of ethyl acetate and hexane (50:50).Cold ethanol was used to wash the reaction mixture upon completion, and the resulting product was dried to obtain acylhydrazone-based isatin derivatives (76-96% yield).The synthetic scheme of the isatin derivatives is illustrated in Scheme 1.

Parallel artificial membrane permeability assay (PAMPA)
The blood-brain barrier (BBB) permeation abilities of the four lead molecules were analyzed using the PAMPA method 44,45 .The detailed procedure is explained in the Supplementary Data.

Cell culture and treatments
The National Center for Cell Science (NCCS), Pune, India, supplied the SH-SY5Y-human bone marrow neuroblastoma cell line, which was maintained in accordance with the recommended protocol in DMEM-high-glucose medium (Cat No. AL111, Himedia) supplemented with 10% fetal bovine serum (FBS) and a 1% antibiotic-antimycotic solution at 37 °C in a CO 2 incubator.Subcultures were performed every two days.Briefly, 5 × 10 5 cells/mL were cultured on a plate and incubated for 24 h to promote cell attachment and reach the required cell density.Neuroinflammation was induced in the SH-SY5Y cells with 1 ug/mL for 2 h followed by cell treatment with different concentrations of the test molecules (IS6, IS7, IS13, and IS15) and incubation for 24 h.LPS-treated cells served as positive controls, and untreated cells served as controls.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
The cytotoxicity of synthetic compounds IS6, IS7, and IS13 was assessed using the common MTT test using SHSY-5Y cells.The cells were grown in 96-well plates with 20,000 cells per well.Different compound concentrations were applied to the cells, and the treated cells were then left to incubate for 24 h.After applying 50 µL of MTT (0.5 mg/mL) to each well, the plates were incubated for 3 h, then 100 µL of DMSO was added to dissolve the purple formazan crystal, and an absorbance was measured at 570 nm using a microplate reader (ELX-800, BioTek, CA, USA).Compounds' growth inhibitory concentration (IC 50 ) values were computed.

Superoxide dismutase (SOD) activity
Using a superoxide dismutase assay kit (KrishGen Biosystems, India), which measures the concentration of formazan crystals using a colorimetric assay, SOD activity was determined.In this test, a tetrazolium salt was used to detect the superoxide radicals produced by xanthine oxidase and hypoxanthine.After treatment, the medium was removed, centrifuged at 2000 rpm for 5 min at room temperature, and placed on ice.A diluted radical detector and lysed cell supernatant or a standard were applied to each well of a 96-well plate to evaluate SOD activity using an ELISA kit, according to the manufacturer's instructions.The absorbance of the wells was then determined after 5 min at a wavelength of 450 nm using a microplate reader (Safire2, Tecan Group Ltd., Maennedorf, Switzerland).The results were displayed as ng/mL [46][47][48][49] .

Glutathione (GSH) activity
A glutathione assay kit (KrishGen Biosystems) was used to determine the GSH levels.The assay kit was based on the disulfide dimer-oxidized GSH reductase recycling method for 5,5'-dithiobis-2-(nitrobenzoic acid) (DTNB).After treatment, adherent cells were removed by scraping the media from the wells.Following suspension in 50 mM phosphate solution (0.5 mL) with a pH of 6.5 and 1 mM ethylenediaminetetraacetic acid, the cells were chilled.The lysed cell supernatant was used to test GSH levels using an ELISA kit.The absorbance of the yellow product was determined at a wavelength of 450 nm.The total GSH activity was estimated using a GSH standard curve.The results were obtained as ng/mL [46][47][48][49]  When GPx activity was rate-limiting, the rate of decline in A450 was directly correlated with GPx activity.Following treatment, adherent cells were removed from the wells, suspended in cold PBS, sonicated, and frozen.In accordance with the ELISA kit's instructions, the lysed cell supernatant, or standard, was applied to all 96 wells of a plate together with a diluted radical detector to assay the activity of GPx.A microplate reader was used to measure the absorbance of the wells after 5 min [46][47][48][49] .Results were obtained in ng/mL.

Catalase (CAT) activity
CAT activity was determined according to Aebi 50 .A human CAT ELISA kit was purchased commercially (Krish-Gen Biosystems).Following treatment, the adherent cells were scraped off, suspended in cold PBS, sonicated, and placed on ice.The medium was then removed from each well.The 3 mL CAT assay combination comprised of extract (0.05 mL), phosphate buffer (1.5 mL, 100 mM buffer, pH 7.0), H 2 O 2 (0.5 mL), and distilled water (0.95 mL).The absorbance decreased at 450 nm.CAT activity was reported in terms of ng/mL of H 2 O 2 oxidized per min per gram [46][47][48][49] .

ROS assays
The OxiSelect Intracellular ROS Assay Kit (Cell Biolabs Inc., San Diego, CA, USA) was used to quantify the levels of the fluorescent probe 20,70-dichlorodihydrofluorescin diacetate (DCFH-DA).In a microplate reader, fluorescence was measured using excitation and emission filters at wavelengths of 488 and 535 nm, respectively 51 .

IL-6, TNF-α, and NF-kB expression
TNF-α, IL-6, and NF-kB expression in cell lysates was assessed using the respective antibodies (PerCP-Cy5.5, PE, and p65-FITC) according to the manufacturer's protocol.Briefly, the spent medium was aspirated, and the cells were treated with LPS (1 µg/mL) for 2 h.Then, the required concentrations of experimental compounds and controls were added and incubated for 24 h.The cells were harvested into polystyrene tubes and centrifuged at 25 °C, washed with PBS, and 70% cold ethanol was added drop wise to create a cell pellet while vortexing.The mixture was then incubated at − 20 °C.The cells were pelleted at a high speed, washed twice with PBS, antibodies added (10 µL), mixed thoroughly, and incubated for 30 min in the dark at 20-25 °C.PBS (500 µL) was added and mixed thoroughly, and the reaction was analyzed using BD FACS-Cell Quest pro software [46][47][48][49]52 .

Statistical analysis
Statistical significance was determined by one-way ANOVA followed by Dunnett-t test using Graph Pad Prism Version 8.0.2.

Molecular docking
The Schrödinger suite 53 was used to perform the molecular docking investigation of IS3, IS6, IS7 and IS13, and IS15.The human MAO-A (hMAO-A, 2Z5Z) and MAO-B (hMAO-B, 2V5Z) X-ray solved structure was obtained from the Protein Data Bank 9,54 .The both crystal structures were improved and optimized using the protein preparation wizard included in the Schrödinger suite, which performed energy minimization, hydrogen atom addition, protonation-state correction, and protonation-state addition.The LigPrep tool was used to construct the ligand structures.The co-crystallized ligands served as the automated center of the grid box.For docking simulations, the force Field OPLS4 default settings and extra precision (XP) docking protocol default settings were used 55,56 .

Molecular dynamic simulation
Schrödinger LLC's Desmond simulation program was used to run the molecular dynamics (MD) simulations 52 .
The protein-ligand combination was initially created for the Desmond system builder panel using compound IS7 against MAO-B in an aqueous solvent system.For complete protein-ligand simulations and stability trajectory analysis (RMSD, RMSF, and protein-ligand contact), the simulation parameters were 100 ns at 300 K, 1.01325 bar pressure, and 1000 frames 55,56 .

MM-GBSA
The Generalized Born and Surface Area (MM-GBSA) solvation technique in Molecular Mechanics was utilized to compute the free binding energies of the ligands to the proteins.In this case, we used several postures from MD simulations of the docked complex to evaluate macromolecular stability and protein-ligand binding affinity.The free energy was calculated using the following formula at the post-processing stage that comes after the MD studies.
The contributions of the internal, electrostatic, and van der Waals energies to molecular mechanics are denoted by the symbols E int , E ele , and E vdw , respectively.Within the equation, the free energy contributions of the polar and non-polar solvation systems are denoted by G pol and G np , respectively.S is an estimate of the entropy, and T is the absolute temperature.The following formula was used to estimate the binding free energy, or ΔG Bind, between the ligand and the protein.
The protein, ligand, and protein-ligand complex are denoted by the letters P, L, and PL, respectively.The equation above expressed the free energy for each of these entities.We used the solvated systems that we obtained prior to performing MD calculations to determine free binding energies.In this case, solvent molecules more than 5 Å away from the bound ligand were replaced with an implicit model via the GB approach, in their postprocessing stages 57,58 .

Synthesis
The target molecules were synthesized in two steps.In the first step, an intermediate acylhydrazide molecule was synthesized by reacting benzoic acid with hydrazine hydrate.This intermediate was then reacted with isatin and halogenated substituted isatins to obtain the final molecules (substituted acylhydrazone-based isatin derivatives: (IS1-IS16) via an acid-catalyzed nucleophilic addition reaction.All the procedures were performed using the microwave reactor.The structures of all synthesized compounds were confirmed by 1 H and 13 C nuclear magnetic resonance ((Bruker Advance Neo 400 MHz NMR spectrometer).The de-shielded protons in all compounds were NH atoms from isatin, and the hydrazone linker exhibited ranges of 11.5-11.0δ and 12.50-14.0δ, respectively.Sharp de-shielded Sp 2 carbonyl carbons of the isatin and hydrazone linkers were observed at 163.60 δ and 141.10 δ, respectively (Supporting Information Figs.S1-S48).
In MAO-A inhibition, compound IS15 (-Cl in the A-ring and -Br in the B-ring) was the highest (IC 50 = 1.852 μM) (Table 1, Fig. S53) and showed 11.94-times higher MAO-A inhibition than IS13 (-Cl in the A-ring and -H in the B-ring), and 10.35-times higher than IS7 (-H in the A-ring and -Br in the B-ring).This indicates that the -Cl substituent in the A-ring contributed to an increase in MAO-A inhibition (Table 1, Fig. 3).These results suggest that compounds IS6, IS7, and IS13 are potent selective MAO-B inhibitors and that compound IS15 is a selective MAO-A inhibitor.

Reversibility studies
Reversibility tests were performed using the dialysis method.In this study, the concentration of compound IS15 used for MAO-A was 1.5 × that of the IC 50 (3.00μM), and those of compounds IS6, IS7, and IS13 used for MAO-B were 1.5 × that of the IC 50 (0.18, 0.12, and 0.15 μM, respectively).Recovery patterns were compared using undialyzed (A U ) and dialyzed (A D ) relative activity after 30 min of pre-incubation.For MAO-A inhibition, compound IS15 recovered from 47.16 to 78.73% (Fig. 4).The recovery of the compound was similar to that of toloxatone (from 33.76 to 87.22%), and it could be distinguished from clorgyline (from 32.32 to 39.23%).For MAO-B inhibition, compounds IS6, IS7, and IS13 recovered from 42.81 to 79.52%, 28.65-72.89%,and 31.45-80.12%,respectively (Fig. 5).The recovery values of the compounds were similar to those of lazabemide (from 41.

Enzyme kinetics
The enzyme kinetics and inhibition types were analyzed at five substrate concentrations and three inhibitor concentrations.In the LB plot, IS15 showed was a competitive MAO-A inhibitor (Fig. 6A), and the secondary plot revealed that the K i value was 1.004 ± 0.171 μM (Fig. 6B).In contrast, IS6, IS7, and IS13 LB plots indicated competitive MAO-B inhibitors (Fig. 7A, C, and E), and the secondary plots showed that their K i values were 0.068 ± 0.022, 0.044 ± 0.002, and 0.061 ± 0.001 μM, respectively (Fig. 7B, D, and F).The K i value of the inhibitor was calculated by the secondary plot constructed with each slope vs. inhibitor concentration in LB plot.The minus value of X-axis of the plot means − K i .Though IS6 and IS7 were not exactly intercepted on one point of Y-axis, V max values in the presence of the inhibitors were almost same within the experimental error range, indicating both also were competitive inhibitors.In the presence of the inhibitors IS6, IS7, IS13, and IS15, K m values were increased and V max values were the same as the control.These results suggest that IS15 is a competitive MAO-A inhibitor, whereas IS6, IS7, and IS13 are competitive MAO-B inhibitors.

PAMPA assay
The PAMPA assay demonstrated that isatin-based hydrazone derivatives (IS6, IS7, IS13, and IS15) had high permeability and CNS bioavailability, with Pe values of > 4.00 × 10 -6 cm/s (Table 2).Brain penetration is crucial for the efficient administration of CNS medication 61 .The effective permeability of the chemical and the equation were used to calculate the penetration rate (Log Pe).A compound is categorized as potentially permeable (CNS+), if its Pe value is > 4.00 × 10 -6 cm/s, and perhaps non-BBB permeable (CNS-), if < 2.00 × 10 -6 cm/s.This study showed that while halogenated isatin has BBB permeability, the substitution of the phenyl ring results in greater penetration.Chloro substitution resulted in higher BBB permeability, as revealed in this study.

MTT, ROS, anti-oxidant, and anti-inflammatory characteristics of the cell line-based assay
Cytotoxic activity of IS6, IS7, and IS13 was tested by MTT assay using the human neuroblastoma SH-SY5Y cells.
The cytotoxicity level was assessed by how well the live cells converted the tetrazolium dye into formazan crystals, with the untreated cells serving as the control group.IS6, IS7, and IS13 remarkably decreased cell viability at 100 μM.However, IC 50 values of IS6, IS7, and 1S13 were 75.72, 97.15, and 85.30 μM, respectively, indicating that these were cell-proliferative and non-cytotoxic in nature or working low concentration to SH-SY5Y cells (Fig. 8).
The preliminary in vitro neuroprotective activity against LPS-induced inflammatory events was tested using the synthesized IS6, IS7, IS13, and IS15 in SH-SY5Y neuroblastoma cell lines.Cell viability assays and ELISA measurements of the intracellular pro-inflammatory cytokines TNF-alpha, IL-6, and NF-kB were used to evaluate neuroprotective activity.A common technique is to incubate SH-SY5Y cell lines with LPS (10 ng/mL) in www.nature.com/scientificreports/minimum necessary medium at 37 °C for 24 h to induce neuroinflammation.We previously discovered that a 2 h incubation period with 1 ug/ml of LPS is adequate to trigger inflammatory responses.LPS treatment significantly raised the levels of IL-6, TNF-alpha, and NF-kB in LPS-intoxicated SH-SY5Y cell lines compared to control SH-SY5Y cell lines, demonstrating the magnitude of inflammatory reactions mediated by LPS toxicity (Fig. 9).IS6, IS7, IS13, and IS15 pretreatment significantly (p < 0.0001) reduced TNFalpha (Fig. 9A), IL-6 (Fig. 9B), and NF-kB (Fig. 9C) levels in comparison to the LPS-treated group, demonstrating the anti-inflammatory potential of isatin derivatives (Figs.S49, S50).Intriguingly, in LPS-treated cell lines, IS6 and IS7 significantly decreased the levels of TNF-alpha and IL-6 compared to IS13 and IS15.When compared to IS6 and IS7, IS15 and IS13 have significantly lower NF-kB expression.The lead compounds confirmed the Table 2. Blood-brain barrier (BBB) assay of key compounds of isatin-based hydrazone derivatives by the parallel artificial membrane permeability assay (PAMPA) method.Pe (10 −6 cm/s) > 4.00: CNS + (high permeation); Pe (10 −6 cm/s) < 2.00: CNS − (low permeation); Pe (10 −6 cm/s) from 2.00 to 4.00: CNS ± (BBB permeation uncertain).

Compound
Experimental Pe (× 10  anti-inflammatory potential of all the compounds in the human neuroblastoma model by inhibiting LPS-induced pro-inflammatory cytokine expression (IL-6, TNF-alpha, and NF-kB).
To further confirm the neuroprotective and anti-oxidant effects of the lead compounds, the effect of the lead compounds on decrease of ROS production using the LPS-treated SH-SY5Y cells (Fig. 10).The compounds IS6, IS7, IS13, and IS15 significantly inhibited (p < 0.0001) 2′,7′-dichlorofluorescin (DCF) expression in the LPSinduced model compared to cells treated with LPS alone.The maximum concentration of test compounds that  significantly inhibited LPS inflammation was considered in the ROS study.The lead compounds, IS15 and IS7, at 10 µM/mL concentrations, exhibited an effective DCF intensity decrease compared to the LPS-induced cells, whereas IS6 and IS13, at 10 µM concentrations, exhibited moderate DCF intensity suppression (Fig. S51).LPS alone induced 65% of DCF expression.The cellular anti-oxidant assay results suggested that the lead compounds showed significant neuroprotective activity by enhancing the cellular oxidant enzymes CAT, GPx, GSH, and SOD (Figs. 11, 12, 13 and 14) in the LPS-induced model, while cells treated with LPS alone effectively expressed SOD, CAT, GSH, and GPx activities.The obtained values confirmed the promising neuroprotective and neuroinflammatory activity of IS6, IS7, IS13, and IS15 compounds (at 10 µM/mL) (p < 0.0001) in relation to the neuroprotective effect in the LPS-induced human neuroblastoma model by enhancing the enzyme activity and inhibiting the oxidative stress-induced apoptosis caused by LPS by determining DCF intensity.

Molecular dynamic simulation
Desmond MD simulations were used to follow the binding mode of IS7 in the inhibitor-binding cavity (IBC) of MAO-B.Protein C-alpha and ligand were tracked within an acceptable range for a long simulation duration (100 ns) according to root mean square deviation (RMSD) analysis.In contrast to the protein RMSD, the ligand (red) RMSD remained steady after 25 ns.The protein RMSD ranged between 1.2 and 3.6 Å with an average of 2.54 ± 0.01 Å (Fig. 17A).The protein-specific RMSD for the simulation was constant, with the exception of a slight variation, reaching a maximum of 3.6 Å at 68-70 ns, where after it stabilized.The simulation evaluated the flexibility of the protein system by computing the RMSF for each amino acid residue of the protein.The 480-498 residues of MAO-B showed a larger fluctuation.The atoms in the benzoyl ring of the RMSF ligand (Fig. 17B) showed slight fluctuations during the binding process.The 21 amino acid residues that interacted with the ligand were Tyr60 (0.541 Å),Gly101 (1.13 Å), Pro102(1.117Å), Pro104 (0.981 Å), Trp119 (1.15 Å), Leu167 (0.955 Å), Phe168 (0.892 Å), Leu171 (0.638 Å), Cys172 (0.706 Å), Ile198 (0.689 Å), Ile199 (0.833 Å), Ser200 (0.88 Å), Thr201 (0.94 Å), Gln206 (0.618 Å), Ile316 (0.604 Å), Tyr326 (0.544 Å), Leu328 (0.572 Å), Met341 (0.493 Å), Phe343 (0.642 Å), Tyr398 (0.97 Å), and Tyr435 (0.497 Å).Hydrogen bonds, hydrophobic contacts, and water bridges were identified in the interaction histograms of IS7 and MAO-B (Fig. 17C and D).Over a trajectory of 100 ns, the number of individual interactions between the amino acids and ligand was normalized.Several significant amino acids, including Tyr326 (hydrogen bond, water bridge, and hydrophobic), Tyr398 (hydrogen bond), Leu171 (hydrophobic), Cys172 (hydrogen bond and water bridge), and Ile199 (water bridge and hydrophobic), interact with IS7.The measured fraction of interactions with Tyr326 was > 1.0.As previously observed 63,64 , the hydrophobic interaction of Tyr326 at the active site of MAO-B was significant.Figure 17C and D depict hydrogen bonding, water bridges, and hydrophobic stability in the ligand-protein complexes.Cys172 forms an 86% hydrogen bond with the carbonyl and NH atoms in the linker between the isatin and benzoyl rings.Tyr398 contributed 49% via hydrogen bonding with the NH atom of the isatin ring.With carbonyl and water molecules, Tyr326 is a 33% active participant in hydrogen bonding.Overall, it is estimated from the trajectory analysis and full MD simulation that the lead compound IS7 will inhibit MAO-B.

MM-GBSA
From their MD simulation frames, the free binding energy was estimated for the best molecule IS7 with the highest docking energy and activity value prediction.Total average energies of ΔG Bind, ΔG Bind H-bond, ΔG Bind Lipo, and ΔG Bind vdW was − 190.04, − 12.26, − 45.94, − 140.23 for 0-100 ns MD snapshot, respectively.Across all interactions, the ΔG Bind vdW and ΔG Bind Lipo energies exerted the most significant impact on the average binding energy (Table 4).
The values ΔG Bind vdW for the interactions of IS7 with protein complexes indicated the presence of stable van der Waals interaction with amino acid residues.Consequently, the MM-GBSA calculations, derived from MD simulation trajectories, aligned well with the binding energies computed from the docking results.The molecule exhibited very low free binding energy, indicating its higher binding affinity towards the receptor.Consequently, it can be inferred that IS7 compound exhibited a strong affinity for the MAO-B protein.

Conclusion
We synthesized acylhydrazone-based isatin compounds and evaluated their ability to inhibit MAOs.IS15 was a potent competitive reversible MAO-A inhibitor, whereas IS6, IS7, and IS13 were potent competitive reversible and selective MAO-B inhibitors.A CNS permeability study using a PAMPA assay revealed that the lead compounds were BBB-permeable.The lead compounds also exhibit non-cytotoxic, neuroprotective and antiinflammatory effects.The lead compounds (at a concentration of 10 µM/mL) effectively reduced DCF intensity.Additionally, a docking analysis of MAO-B and IS7 revealed the stability of the complex due to the pi-pi stacking of Tyr326.The Cys172 residue participated in the interaction with the ligand at 86% during dynamic examination.Finally, MM-GBSA energy binding revealed that IS7 provided strong stability to MAO-B protein.Overall, the results of this investigation suggest that the lead compounds, IS7, IS6, IS13, and IS15, may be viable therapeutic agents for the treatment of neurological disorders such as PD.

Figure 1 .
Figure1.Base structure of isatin and their derivatives for FDA-approved drugs.

Figure 4 .
Figure 4. Recovery of MAO-A inhibition by IS15 using dialysis experiments.
77.71%) and could be distinguished from those of pargyline (from 41.04 to 34.34%).These results indicate that IS15 is a reversible inhibitor of MAO-A, whereas IS6, IS7, and IS15 are reversible inhibitors of MAO-B.

Figure 6 .
Figure 6.Lineweaver-Burk (LB) plots for MAO-A inhibition by IS15 (A) and their respective secondary plots (B) of the slopes vs. inhibitor concentrations.

Figure 17 .
Figure 17.Desmond's MD simulation analysis of the IS7-MAO-B complex.(A) Root mean square deviation (RMSD) (protein and IS7 RMSD are shown in blue and red, respectively).(B) Individual RMSF for proteins' amino acids.(C) Diagram of the 2-D Interaction.(D) Protein-ligand contacts with number of specific contacts of amino acids with IS7.
. KrishGen Biosystems) was used to evaluate the GPx activity.The kit uses a colorimetric assay to determine the quantity of GPx.Glutathione reductase (GR) mediates GPx activity Oxidized glutathione (GSSG) is produced via hydroperoxide reduction by GPx.This glutathione is recycled back to its reduced state by GR and NADPH.The NADPH to NADP + oxidation was accompanied by a decrease in absorbance at 450 nm (A450).